The cloning of each of the pharmacologically identified types of opioid receptors (i.e., delta, mu and kappa) has dramatically altered the nature of the questions that can be addressed in the problem of the mismatch between opioid receptors and their endogenous ligands. Two questions that arise are: "Do the cloned receptors account for all of the opioid receptors in the nervous system?" and "Do the identified endogenous ligands represent the complete set of ligands available for action at opioid receptors?" With the successful production of a mu opioid knockout mouse and the isolation and characterization of a new family of neuropeptides (the endomorphins) that are active in mu receptors, these questions can be addressed in novel ways. It is with this in mind that we propose experiments that will contribute to the realization of the following specific aims: 1) Determine if the cloned mu receptor, MOR1, is the mu receptor responsible for presynaptic inhibition of norepinephrine release from axons of locus coeruleus neurons. 2) Determine the biosynthetic precursor responsible for synthesis of endomorphin, and the regional distribution of cells that express the transcript for endomorphin and its biologically active products. 3) Determine the spatial relationship between the endomorphin peptides and MOR1. 4) Determine if the expression of endomorphin is altered in mice that lack the apparent cognate receptor (MOR1). These aims will be accomplished by several different types of experiments. Synaptosomal preparations of the forebrain of wild type and mu knockout mice will help to elucidate the extend to which MOR1 is responsible for the presynaptic inhibition of norepinephrine release. Also, proposed are expression cloning experiments to isolate the biosynthetic precursor of the endomorphins, followed by in situ hybridization studies. Finally, we propose the development of specific antisera to the endomorphins and subsequent two-color immunofluorescent experiments in wild type and knockout mice to determine the extent of match between the cloned mu receptor and this new family of endogenous ligands.